History

Gossypol was first identified as an antifertility agent as a result of epidemiologic studies conducted in China during the 1950s.

Investigators had been puzzled by the extremely low birth rates in a particular geographic region.

The men had very low sperm counts and many women had amenorrhea. Eventually the phenomenon was attributed to the exclusive use of crude cottonseed oil for cooking. Further investigation revealed that the antifertility component was gossypol, a potentially toxic phenolic pigment found in the seed, stem, and root of the cotton plant. Male “pill” blocks sperm enzyme. 1981

Sources

Gossypol is a natural product that can be made synthetically Gossypol, a pigment of cottonseed. 1960
or produced inexpensively on a very large scale by the extraction of cottonseed. Estimates have placed potential US gossypol production at more than 50,000 tons per year, all as a by-product of cottonseed oil production. Cotton represents the most well-known source of this compound. The seeds of the Gossypium species vary widely in gossypol content, with levels ranging from 0.13% to 6.6%.

Biosynthesis

Gossypol is a terpenoid aldehyde, which is formed metabolically through acetate via the isoprenoid pathway. Sesquiterpene dimer undergoes a radical coupling reaction in order to form gossypol Biosynthesis of Gossypol. 1970

The biosynthesis of gossypol is summarized in figure below.

The biosynthesis begins with cadinyl carbocation a sesquiterpene precursor derivated from geranyl pyrophosphate. It is oxidized to (+) delta cadinene by the enzyme (+) deltha cadinene synthase. Subsequently, the NADPH dependent enzime (+) delta cadinene 8- hydroxylase oxidized the (+) delta cadinene to 8 hydroxy delta cadinene . This compound goes through various oxidative processes to make deoxyhemigossypol which is oxidized by one-electron into hemigossypol and then undergoes a phenolic oxidative coupling ortho to the phenol groups to form gossypol. The coupling is catalyzed by an H2O2 dependent peroxidise enzyme which results in the formation of the racemic mixture. Medicinal Natural Product: A Biosynthetic approach. Third ed.; 2008]

Gossypol Uses and Pharmacology

Contraception

Gossypol is a nonsteroidal compound that inhibits sperm production and motility in a variety of male animals and in humans. It does not affect sex hormone levels or libido, and its mechanism is distinct from that of steroidal oral contraceptives used by women.
Gossypol exerts its contraceptive action by inhibiting an enzyme that plays a crucial role in energy metabolism in sperm and spermatogenic cells. The target enzyme, lactate dehydrogenase X, is found only in sperm and male gonadal cells. It is involved in glycolysis and plays a role in inducing mitochondria to produce energy. LDH-X, an isoenzyme of lactate dehydrogenase specific for germinal epithelium activity, has been measured in the seminal plasma of infertile subjects whose infertility had different origins. In the same samples, seminal transferrin, an index of Sertoli cell function, was also measured. In this investigation, seminal LDH-X was not detectable in the vasectomized subjects, in patients with azoospermia due to seminiferous tubular damage, nor in patients who showed a marked decrease in sperm concentration (less than 106/ml). In oligozoospermic patients (sperm concentration less than 20 × 106/ml) seminal LDH-X levels were reduced to about one-third of those found in normal controls. Seminal LDH-X levels correlated (r = 0.7237) with total sperm count better than seminal transferrin levels (r = 0.5511), while no correlation was found between these two biochemical parameters and sperm motility, viability and morphology. To study their spontaneous variations with time, LDH-X and transferrin were also measured in semen specimens collected monthly from five healthy men, over 1 year. In these samples (N = 60), sperm count variability (43.1%, calculated in terms of the coefficient of variation), was similar to that of LDH-X (40.4%) and higher than for transferrin (23.0%) [6] [Orlando C, et al. Measurement of Seminal LDH-X and Transferrin in Normal and Infertile Men. Journal of Andrology, Vol. 9, No. 3, May/June 1988]
A variety of lactate dehydrogenases are found throughout the body and gossypol exerts a degree of inhibition on many of these. However, the drug exhibits its greatest inhibitory effect on lactate dehydrogenase X.

Male contraception

Preclinical studies

Gossypol has been shown to be an active antifertility agent in male hamsters and rats [7] [Waller DP, Fong HH, Cordell GA, Soejarto DD. Antifertility effects of gossypol and its impurities on male hamsters. Contraception . 1981;23:653-660]. Sperm recovered from the epididymis of rats and hamsters treated with gossypol for as little as 6 weeks were immotile, with detached heads or tails [8] [Chang MC, Gu Z, Saksena SK. Effects of Gossypol on the fertility of male rats, hamsters and rabbits. Contraception. 1980;21:461-469] Gossypol does not demonstrate estrogenic or androgenic activity but it can potentiate the androgenicity of methyltestosterone [9] [Touvay C, Vilain B, Sirois P, Soufir M, Braquet P. Gossypol: a potent inhibitor of PAF-acether- and leukotriene-induced contractions of guinea-pig lung parenchyma strips. J Pharm Pharmacol . 1987;39:454-458]

Clinical data

Large-scale clinical trials in men by Chinese investigators have found gossypol to be orally active and relatively safe and effective. The clinical testing of gossypol began in the early 1970s in China, and to date the drug has been studied extensively in thousands of men. The usual daily dose is 20 mg until the sperm count is reduced below 4million/mL; this usually requires 2 to 3 months of treatment. Maintenance doses of 75 to 100 mg are subsequently taken twice a month. A proposed contraceptive dose is 3 g/man/year.
A comprehensive review of the clinical trials of gossypol has been published [10] [Wu D. An overview of the clinical pharmacology and therapeutic potential of gossypol as a male contraceptive agent and in gynaecological disease. Drugs . 1989;38:333-341]. Clinical trials have found the drug's antifertility effect to be more than 99%. Sperm counts usually return to normal within 3 months after termination of therapy, and men treated with gossypol have fathered normal children. However, long-term follow-up studies indicate that inhibition of spermatogenesis may continue following discontinuation in up to 20% of men after 2 years. Spermatogenesis does not return to normal in some men. Concerns regarding the lack of predictable, reversible effects have delayed the further clinical development of gossypol in Western countries.

Female contraception

Preclinical studies

Although gossypol is generally considered to be a male antifertility agent, it is also effective when given to female animals. The intramuscular injection of gossypol into female rats inhibited implantation and the maintenance of normal pregnancy, most likely by affecting luteinizing hormone levels [11] [Lin YC, Fukaya T, Rikihisa Y, Walton A. Gossypol in female fertility control: ovum implantation and early pregnancy inhibited in rats. Life Sci. 1985;37:39-47]. Gossypol is also an inhibitor of platelet activating factor and leukotrienes [12] [Touvay C, Vilain B, Sirois P, Soufir M, Braquet P. Gossypol: a potent inhibitor of PAF-acether- and leukotriene-induced contractions of guinea-pig lung parenchyma strips. J Pharm Pharmacol . 1987;39:454-458] In addition, gossypol has been evaluated as a topical spermicide. A gelatin coprecipitate of gossypol is an effective spermicide in monkeys [13] [Cameron SM, Waller DP, Zaneveld LJ. Vaginal spermicidal activity of gossypol in the Macaca arctoides . Fertil Steril . 1982;37:273-274]

Clinical data

Gossypol has shown effects that suggest some inhibition of ovarian function and cytotoxicity in endometrial cells [14] [Zu PD, Sun YT, Cheng J, Tian L, Dang MY, Han ML. Electron microscopic observations of the effects of gossypol on the human endometrium. Am J Obstet Gynecol . 1984;149:780-787]. Gossypol has been evaluated as a topical spermicide in humans. Solutions of gossypol or gossypol acetate do not decrease sperm motility. However, a solution of gossypol-PVP coprecipitate completely immobilized all spermatozoa within 3 minutes at a concentration of 5 mg/mL and within 20 seconds at 40 mg/mL. The spermicidal activity of gossypol was found to be equal to or greater than that of the commercially available creams Delfin and Preceptin, as well as Encare Oval vaginal suppositories [15] [Waller DP, Zaneveld LJ, Fong HH. In vitro spermicidal activity of gossypol. Contraception. 1980;22:183-187]. A gossypol-containing gel decreased the number and rapidly immobilized sperm when tested in healthy women. [16] [Ratsula K, Haukkamaa M, Wichmann K, Luukkainen T. Vaginal contraception with gossypol: a clinical study. Contraception . 1983;27:571-576]. The topical use of gossypol has been recommended because it has low systemic toxicity, it acts in micromolar concentrations, and it is effective in the presence of cervical mucus. [17] [Poso H, Wichmann K, Janne J, Luukkainen T. Gossypol, a powerful inhibitor of human spermatozoal metabolism. Lancet . 1980;1:885-886]

Other application.

Gossypol and its derivatives are active against the HIV virus 15 and the herpes simplex virus type 2. [35] [Lin TS, Schinazi. Selective inhibition of human immunodeficiency virus type 1replication by the (-) but not the (+) enantiomer of gossypol. Antimicrob Agents Chemother. 1989 Dec;33(12):2149-51]. A trial evaluated the therapeutic efficacy of gossypol for the treatment of metastatic carcinoma of the endometrium or ovary, and as an antiviral and interferon inducer in patients with AIDS [36] [Wu D. An overview of the clinical pharmacology and therapeutic potential of gossypol as a male contraceptive agent and in gynaecological disease. Drugs . 1989;38:333-341]

At low doses (30 mg/kg), the drug reduces 65% the tumor size and induces tumor mortality (8%) in nude-mice SW-13 adrenocortical carcinoma [24] [Wu, Y.W., Chik, C. L., Knazek, R. A., An in vitro and in vivo study of antitumor effects of gossypol on human SW-13 adrenocortical carcinoma, Cancer Res. 1989, 49, 3754 –3758.] . Furthermore, in the PC-3 prostate tumour xenograft study, (-)-gossypol (> 5 mg kg-1) given once a day for 7 days significantly inhibited tumour growth in a dose-dependent manner. Immunohistochemical analysis revealed that (-)-gossypol enhanced caspase-3 and caspase-8 expression and decreased the expression of PCNA, Bcl-2 and CD31 in tumour tissues. It suggested that cell apoptosis and inhibition of angiogenesis might contribute to the anticancer action of (-)-gossypol [25] [Zhang XQ, et al. Inhibition of proliferation of prostate cancer cell line, PC-3, in vitro and in vivo using (-)-gossypol. Asian J Androl. 2010 May;12(3):390-9]. Both racemic form and (-)-enanthiomer (AT-101, Ascenta Therapeutics http://www.ascenta.com/) resulted in a significant cytotoxicity and induced apoptosis in OVCAR-3 cells. This effect was observed in a dose- and time dependent manner. However, AT-101 was much more potent. In addition, the treatment of 10 microM of racemic gossypol alone and 3 microM of AT-101 alone resulted in significant down-regulation (>or= 3 fold) in mRNA levels of some pivotal angiogenic molecules in OVCAR-3, but altered gene profiles were different by the treatment of each enantiomer [26] [Varol U, et al. The effect of racemic gossypol and at-101 on angiogenic profile of ovcar-3 cells: a preliminary molecular framework for gossypol enantiomers. Exp Oncol. 2009 Dec;31(4):220-5]. More recently it was demonstrated that gossypol is able to inhibit Bcl-2 family antiapoptotic proteins [27] [Zhang M, et al. Molecular mechanism of gossypol-induced cell growth inhibition and cell death of HT-29 human colon carcinoma cells. Biochem Pharmacol. 2003 Jul 1;66(1):93-103] [28]

Toxicology

Gossypol inhibits malate dehydrogenase in animals and glutathione-S-transferase in both animals and humans. This latter enzyme is involved in the detoxification of potentially toxic and carcinogenic compounds. Gossypol has not been found to be mutagenic when tested in the Ames salmonella microsome test, [37] [de Peyster A, Wang YY. Gossypol—proposed contraceptive for men passes the Ames test. N Engl J Med . 1979;301:275-276] but questions still remain about its genotoxic characteristics [38] [de Peyster A, Wang YY. Genetic toxicity studies of gossypol. Mutat Res . 1993;297:293-312] Gossypol toxicity is a potential veterinary problem, and as little as 200 parts per million of free gossypol could kill a calf [38]. Nonruminant animals are more sensitive to the toxic effects of gossypol than ruminants [39] [Randel RD, Chase CC Jr, Wyse SJ. Effects of gossypol and cottonseed products on reproduction of mammals. J Anim Sci . 1992;70:1628-1638] The (-) isomer is active as a male contraceptive whereas toxic symptoms are associated with the (+) isomer.

Clinical data

An open-label, multicenter, phase I/II study of single-agent AT-101 in men with castrate-resistant prostate cancer demonstrated that AT-101 administered at 20 mg/day for 21 of 28 days was well-tolerated. Evidence of single-agent clinical activity was observed with prostate-specific antigen declines in some patients [29] [Liu G, et al. An open-label, multicenter, phase I/II study of single-agent AT-101 in men with castrate-resistant prostate cancer. Clin Cancer Res. 2009 May 1;15(9):3172-6]. Further investigation of AT-101 in prostate cancer is warranted and trials combining AT-101 with androgen deprivation, as well as with docetaxel chemotherapy prednisone are ongoing [30] [MacVicar G, et al. An open-label, multicenter, phase I/II study of AT-101 in combination with docetaxel (D) and prednisone (P) in men with castrate resistant prostate cancer (CRPC). Abstract #5062; Journal of Clinical Oncology, 2009 ASCO Annual Meeting Proceedings] [31] [Poiesz B, et al. Preliminary report of an open-label, multicenter, phase I/II study of AT-101 in combination with docetaxel (D) and prednisone (P) in men with docetaxel refractory prostate cancer. Abstract #5145; Journal of Clinical Oncology, 2009 ASCO]
In NABTT-0702 a phase II study of R-(-)-gossypol (AT-101) in recurrent glioblastoma multiforme 65 patients were enrolled with a median age of 59 (range 34–79) and KPS of 80 (range 60–100). Grade 3 or greater adverse events possibly or probably related to AT-101 included fatigue (n = 2), ileus (n = 1), elevated cardiac troponin T levels (n = 1), elevated GGT (n = 1), and thrombocytopenia (n = 1) were registered. Response data is available for 43 patients. OS determination is ongoing. Seven patients (16%) had stable disease as the best response. One partial response (centrally reviewed) was observed. No complete responses were observed. Although treatment is ongoing, two patients are without progression after more than 7 months of therapy. AT-101 is well tolerated and without unique toxicities in patients with recurrent GBM. Further follow-up will determine the impact of AT-101 on OS and whether any tissue correlate is predictive of efficacy. [32] [Fiveash J, et al. NABT-0702: A phase II study of AT-101 in recurrent glioblastoma multiforme (GBM). [Abstract #2010 Journal of Clinical Oncology, 2009 ASCO Annual Meeting Proceedings]. The interim analysis of a phase II trial of AT-101 in patients with recurrent extensive stage small cell lung cancer indicates that AT-101 is well-tolerated but has little activity in recurrent chemosensitive SCLC [33] [J Clin Oncol 28, 2010]

In phase I trials, gossypol decreased glial and adrenal tumor size by 10–50% [34] [Bushunow, P., Reidenberg, M. M., Wasenko, J., Lorenzo, B.et al., Gossypol treatment of recurrent adult malignant gliomas, J. Neurooncol. 1999, 43, 79–86]. In addition gossypol has been investigated for the treatment of uterine myoma, endometriosis, and dysfunctional uterine bleeding.
Other application.
Gossypol and its derivatives are active against the HIV virus 15 and the herpes simplex virus type 2. [35] [Lin TS, Schinazi. Selective inhibition of human immunodeficiency virus type 1replication by the (-) but not the (+) enantiomer of gossypol. Antimicrob Agents Chemother. 1989 Dec;33(12):2149-51]. A trial evaluated the therapeutic efficacy of gossypol for the treatment of metastatic carcinoma of the endometrium or ovary, and as an antiviral and interferon inducer in patients with AIDS [36] [Wu D. An overview of the clinical pharmacology and therapeutic potential of gossypol as a male contraceptive agent and in gynaecological disease. Drugs . 1989;38:333-341]